PHARMACOLOGICAL AND IMMUNOLOGICAL CHARACTERIZATION OF N-METHYL-D-ASPARTATE RECEPTORS IN HUMAN NT2-N NEURONS

NT2 cells are a clonal line of human teratocarcinoma cells that exhibi t N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity after t erminal differentiation into NT2-N neurons. In this study, we used mod ulation of glutamate excitotoxicity to characterize the pharmacologica l properties and specific antibodies to determine the individual subun its of NMDA receptors expressed by NT2-N neurons. The glycine site ant agonist 7-chlorokynurenic acid completely blocked glutamate toxicity i n a dose-dependent manner. Histamine and the polyamine agonists spermi ne and spermidine enhanced glutamate toxicity in a dose-dependent mann er consistent with expression of an NR1-NR2B combination of subunits. The efficacy of polyamine agonists suggests the expression of one or m ore splice variants of the NR1 subunit that lack the putative surface loop encoded by exon 5. Surprisingly, the putative inverse agonists di aminodecane and diaminododecane also enhanced toxicity in a dose-depen dent manner. The antagonists arcaine and ifenprodil completely blocked glutamate toxicity in NT2-N cells. The atypical antagonist ifenprodil inhibited toxicity with a uni formly high affinity characteristic of interaction with the NR1-NR2B combination of subunits. Expression of b oth NR1 and NR2 subunits were detected by Western blot analysis. Neith er protein was detectable in undifferentiated cells. In contrast, 70-f old lower levels of the NR2A subunit were detected in both differentia ted and undifferentiated cells. The pharmacological and immunological results indicate that a functional NR1-NR2B combination of subunits is expressed by NT2-N neurons. Despite the immunological detection of NR 2A subunit, no functional combination of NR1 and NR2A subunits could b e demonstrated.